Bioreactor and method of forming complex three-dimensional tissue constructs

ABSTRACT

A bioreactor and method of forming complex three-dimensional tissue constructs in a single culture chamber. The bioreactor and methods may be used to form multi-phasic tissue constructs having tissue formed from multiple cell types in a single culture chamber. The bioreactor includes at least one translation mechanism to facilitate translation of one or more tissue constructs without direct user intervention, thereby providing a closed, sterile environment for complex tissue fabrication. The bioreactor may be used as a stand-alone device or as part of a large-scale system including many bioreactors. The large-scale system may include a perfusion system to monitor and regulate the tissue culture environment.

TECHNICAL FIELD

This invention relates generally to bioreactors, and more particularlyto bioreactors that facilitate the formation of complexthree-dimensional and multi-phasic tissue constructs.

BACKGROUND

While advances in regenerative medicine have flourished, significanthurdles remain with respect to the commercial manufacture of consistent,uniform, reproducible tissue constructs. Relatively few products haverealized commercial success as tissue substitutes despite significantadvancements in the field of tissue engineering. Several challengesstill remain for translating tissue-engineering technologies from benchto bedside. In addition to demonstrating clinical effectiveness,cost-effective manufacturing processes that comply with current qualityand safety regulations are desirable. Failure to adequately consider thelikely cost of manufacturing early in process and product developmenthas been recognized as a limiting factor for commercial success oftechnologies in the field. Traditional manual laboratory tissue culturetechniques are not economical at large clinical scales and have inherentvariability and contamination risks. However, due to their widespreaduse, simplicity, minimal development time and low initial costs, theseprocesses are often used to advance engineered tissues into clinicaltrials and the marketplace.

For example, Carticel, available from Aastrom Biosciences, Inc. (AnnArbor, Mich.), is an autologous cell transplantation product used torepair articular cartilage injuries in the knee. Carticel ismanufactured via manual techniques performed by trained technicians in aserologic clean room. Briefly, cells are isolated from the patient andexpanded on tissue culture flasks to a sufficient number necessary forthe desired therapeutic use. This process requires a large number ofmanual labor-intensive manipulation steps. Manual processes such asthis, though perhaps more feasible and economical on a small-scaleduring early process development, are generally regarded as high riskdue to the increased potential for contamination. Additionally, inherentdifferences in processing techniques between individuals, especiallywith technically challenging methods, can lead to processinconsistencies and end product variability. Furthermore, as productiondemand increases, scale-up would require multiple manual processes to beperformed in parallel, thereby requiring additional technicians andgenerating substantial labor costs. As a result, the overall cost ofproduction of such products tends to be high and can limit clinicalsuccess when the cost-benefit of the product is evaluated againstcompeting, and often-simpler therapeutic strategies. Automating manualtissue-culture processes through use of bioreactor systems can provide ameans to standardize culture processes, tightly control cultureenvironments, remove user-dependent operation, and enable cost-effectivetissue manufacturing processes to meet large-scale clinical demand.

Automated bioreactors have previously been used in tissue-engineeringmanufacturing and typically involve streamlining traditional twodimensional cell culture processes (e.g., cell selection, expansion,differentiation, etc.). While important, these cell manufacturingprocesses are often only the starting point for many tissue engineeringstrategies that utilize well characterized cell populations for tissuegrowth. Thus, there is a need to foster automated bioreactor-basedsystems fit for the development of reproducible three dimensionalconstructs. A closed, standardized, and operator-independent bioreactorsystem would have the potential to ensure safety and regulatorycompliance and enable cost-effective, large-scale, in vitro tissueproduction.

Recently, Advance Tissue Science developed an automated system for largescale manufacturing of their human fibroblast derived dermal substitute,Dermagraft (currently manufactured by Organogenesis, Inc. (Canton,Mass.)). Dermagraft consists of living cryopreserved allogeneic dermalfibroblasts seeded onto a bio-absorbable polyglactin mesh scaffold usedfor treatment of chronic skin wounds such as diabetic foot ulcers. Theentire manufacturing process is performed in a closed bioreactor bag,eliminating user-dependent variability and shielding the culture processfrom risk of contamination. Following injection of cells into thesystem, the bioreactor is only externally manipulated, until the graftis thawed and utilized in the operating room.

A single Dermagraft bioreactor bag can be used to manufacture up toeight grafts, each in individual compartments. Additionally, twelve bagscan be attached to an automated perfusion manifold system, standardizingculture parameters and allowing for up to 96 grafts to be made in asingle production run. From a scientific perspective, the ability of anautomated bioreactor system to systematically control and manipulate keyculture parameters is important for providing data sets needed tostandardize production methods and optimize end-product criteria. Thoughinitial development costs of automated bioreactor systems are oftenhigh, the lack of user-dependence and ability to create large productionvolumes can greatly improve the long term cost effectiveness andcommercial viability of the process. The lack of sufficient processcontrol early in the Dermagraft manufacturing process resulted in manydefective products and ultimately contributed to overall high productioncosts. Thus, there is a need for process and manufacturing issues to beaddressed earlier in product life cycle to increase likelihood ofcommercial success and clinical benefit. Failure to addressmanufacturing concerns early can lead to process changes later duringpre-market approval that will likely require revalidation or additionalstudies to ensure safety and efficacy.

Current automated bioreactor systems such as the Dermagraft are productspecific and may pose problems for the fabrication of largerthree-dimensional tissues. Moreover, no bioreactor currently exists forthe fabrication of three-dimensional bone-ligament tissue constructs,such as those described in U.S. Pat. No. 8,764,828, assigned to theApplicant of the present application and hereby incorporated byreference in its entirety. To facilitate the manufacture of a wide-rangeof multi-phasic, three-dimensional tissue constructs, a novel bioreactormust be developed. As with current automated manufacturing systems, thebioreactor design should be user-independent, require only externalmanipulation, minimize contamination risk, and be scalable. It shouldalso recreate the unique multi-step formation, technician-dependentprocesses often required when forming multi-phasic tissue constructs.

Transitioning the laboratory concepts and methods involved with themanufacture of multi-phasic tissue constructs into well-characterizedmedical products and processes is a significant challenge, commonlyunderestimated by researchers in tissue engineering and regenerativemedicine fields. In addition to meeting regulatory requirements andensuring the consistency and safety of the desired product, themanufacturing system must be scalable and cost-effective to beeconomically viable and displace current treatment options. Addressingmanufacturing issues related to scale-out, quality control, andcost-effective production early in the research stage can overcomeproblems that typically slow development, limit investment, and escalatecosts that limit the clinical translation and availability of varioustreatment methods to patients in need.

SUMMARY

According to one embodiment, there is provided a bioreactor for forminga complex three-dimensional tissue construct. The bioreactor comprises afirst substrate for culturing a first cell source and a second substratefor culturing a second cell source. The first substrate and the secondsubstrate comprise two walls of a culture chamber. The bioreactorfurther comprises at least one translation mechanism in the culturechamber extending at least partially between the first substrate and thesecond substrate so that a first tissue construct of the first cellsource cultured on the first substrate or a second tissue construct ofthe second cell source cultured on the second substrate can betranslated via the translation mechanism to form the three-dimensionaltissue construct having the second tissue construct of the second cellsource co-cultured with the first tissue construct of the first cellsource.

According to another embodiment, there is provided a method of forming amulti-phasic tissue construct in a single culture chamber. The methodcomprises the steps of forming a first tissue construct in the culturechamber, adding cells to the culture chamber containing the first tissueconstruct, and culturing the cells to form a second tissue construct inthe culture chamber. The first tissue construct and the second tissueconstruct are formed from different cell types and together comprise themulti-phasic issue construct.

According to another embodiment, there is provided a method of forming acomplex three-dimensional tissue construct in a single culture chamber.The method comprises the steps of adding cells from a first cell sourceto a first substrate in the culture chamber, adding cells from a secondcell source to a second substrate in the culture chamber, culturing thefirst cell source and the second cell source to form a first tissueconstruct and a second tissue construct, translating the first andsecond tissue constructs via one or more translation mechanisms to aco-culture zone between the first and second substrates, and forming thethree-dimensional tissue construct having the second tissue construct ofthe second cell source cultured with the first tissue construct of thefirst cell source.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred exemplary embodiments will hereinafter be described inconjunction with the appended drawings, wherein like designations denotelike elements, and wherein:

FIG. 1 is an exploded view of a bioreactor in accordance with oneembodiment;

FIG. 2 is an image of a bioreactor in accordance with one embodiment;

FIG. 3 is a perspective, exploded view of one embodiment of a componentfor a translation mechanism for a bioreactor;

FIG. 4 is a top view of a bioreactor having a translation mechanism inaccordance with one embodiment;

FIG. 5 illustrates a bioreactor in accordance with one embodiment;

FIG. 6 is a perspective view of a bioreactor detailing the position ofthe translation mechanisms in accordance with one embodiment;

FIG. 7 is an image of an assembly of bioreactors in accordance with oneembodiment;

FIGS. 8-10 show multi-phasic tissue constructs formed in accordance withvarious embodiments;

FIGS. 11A-11E illustrate various steps involved in one embodiment offorming a complex three-dimensional tissue construct;

FIG. 12 is a perspective view of a bioreactor in accordance with oneembodiment;

FIGS. 13A and 13B show various views of the body of the bioreactor shownin FIG. 12;

FIGS. 14A and 14B show various views of a cap that may be used for abioreactor, such as the bioreactor shown in FIG. 12;

FIG. 15 shows translation mechanisms and a substrate that may be usedwith the bioreactor of FIG. 12;

FIGS. 16A-16F illustrate various cell culture surfaces that may be usedwith bioreactor substrates;

FIG. 17 schematically illustrates tissue construct formation in abioreactor having a similar structure to the bioreactor depicted in FIG.12;

FIGS. 18 and 19 schematically represent tissue construct formation thatis possible with the bioreactor shown in FIG. 12 in accordance with twoembodiments;

FIGS. 20A and 20B show various views of a rotary device that may be usedwith the bioreactor shown in FIG. 12;

FIG. 21 illustrates a removing tool that may be used with the bioreactorshown in FIG. 12;

FIG. 22 is an image of a multi-phasic tissue construct formed inaccordance with one embodiment and shown on a tensile test platform;

FIG. 23 is a graph showing stress-strain data of multi-phasic tissueconstructs formed in accordance with various embodiments;

FIGS. 24A-24F show histological staining results of multi-phasic tissueconstructs formed in accordance with one embodiment; and

FIG. 25 illustrates polymerase chain reaction (PCR) data for amulti-phasic tissue construct formed in accordance with one embodiment.

DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

A bioreactor capable of producing complex three-dimensional tissueconstructs, such as the bioreactor described herein, can promote theaccessibility and availability of a diverse array of tissue constructsfor various treatment and research purposes. Complex three-dimensionaltissue constructs can include multiple tissue constructs formed frommultiple cell sources, and more particularly, complex three-dimensionaltissue constructs can include multi-phasic tissue constructs thatinclude multiple tissue constructs cultured from cell sources with oneor more of the cell sources containing different cell types (e.g., boneand ligament cells). The bioreactor can allow for cells of two differentsources to be grown independently and then co-cultured to fabricate ascaffold-free, multi-phasic three-dimensional engineered tissue with twoor more different types of tissue in the final product. The bioreactorcan be a stand-alone culture plate or part of an assembly that comprisesnumerous bioreactors and an environmental control system or perfusionsystem, for example. The bioreactor can allow for the formation ofcontinuous, multi-phasic tissue constructs without any internalmanipulation by the technician, thereby minimizing contamination risk.In addition to minimizing contamination risk, a closed bioreactor systemcan precisely control and maintain the tissue culture environment.Tissue growth and development is highly responsive to environmentalfactors (pH, temperature, nutrient concentration, etc.), and as such,the entire fabrication process can impact the integrity of the finalproduct. Thus, it is desirable if the process maintains a high level ofcontrol to meet the rigorous current good manufacturing practices (CGMP)standards required by the Food and Drug Administration (FDA) to ensuretissue safety and manufacturing consistency (see Chapter 21 of the Codeof Federal Regulations). The bioreactor disclosed herein can facilitatesuch an improved fabrication process, and advantageously, the complexthree-dimensional tissue construct can remain in a closed or partiallyclosed environment until it is ultimately needed for patient orresearcher use. Through the elimination of manual, user-dependentprocesses, the bioreactor and method facilitate an automated system andprocess that can eliminate user variability and that promotes tissueconstruct consistency. Tissue manufacturing times may be reduced, andproduction capacity may be increased. Further, the system may bemodular, capable of incorporating multiple bioreactors into a largercentrally controlled assembly while, in some embodiments, maintaining arelatively small overall volume to minimize the space required tomanufacture the tissue constructs.

FIG. 1 is an exploded view of one embodiment of a bioreactor 10 forforming one or more complex three-dimensional or multi-phasic tissueconstructs, and FIG. 2 is an image of an assembled bioreactor inaccordance with the FIG. 1 embodiment. Bioreactor 10 includes a culturechamber 12; however, it should be appreciated that more culture chambersmay be included depending on the particular bioreactor design. Thebioreactor includes a first substrate 14 for culturing a first cellsource and a second substrate 16 for culturing a second cell source. Thefirst substrate 14 and the second substrate 16 constitute two walls ofthe culture chamber 12, and in one embodiment, the substrates areconstructed from a sterile polystyrene culture surface that may becoated with growth factors or cell adhesion proteins such as laminin.The addition of cell adhesion proteins can facilitate adherent cellculturing. The bioreactor further includes at least one translationmechanism 18 extending between the first substrate 14 and the secondsubstrate 16, and in this embodiment, there are two sutures in eachculture chamber 12 that constitute the translation mechanism 18.Translation component 20 may be included to help facilitate movement ofthe translation mechanism 18. Other translation mechanism embodimentsare described in more detail below. A non-adherent barrier 22 may alsobe included to at least partially separate the culture chamber 12 intotwo culture vessels 13. Multiple culture vessels allow for the formationof multiple monolayers of various shapes and sizes. For example, twotissue constructs of the first tissue type may be formed, and then thebioreactor may be flipped, inverted, turned, or rotated so as to allowthe second tissue construct of a second tissue type to form between thetwo tissue constructs of the first tissue type. If desired, thenon-adherent barrier 22 may be made from silicone, Teflon, or anothermaterial that does not allow for significant cell adhesion.

The bioreactor may further include ports 24 for exchanging various typesof culture media. In the illustrated embodiment, ports 24 are locatedwithin a connecting plate 26 that is situated between the firstsubstrate 14 and the second substrate 16. More or less ports may beincluded and may be integrated with other components of the bioreactor,depending on the particular design requisites. In one embodiment, thebioreactor includes a perfusion system (not shown) that is incommunication with the ports 24 for temperature and/or fluid exchange,for example. Any standard perfusion system and control system can beused to monitor and control the conditions in and out of the bioreactor.In a preferred embodiment, the perfusion system includes pH,temperature, carbon dioxide, and oxygen level monitoring. A computercontrolled system may run pumps that add and subtract culture media andrecord measurements from various sensors. The bioreactor 10 may beclosed, using a perfusion system to control the internal environment, orit may be partially closed, leaving the ports 24 open to the externalenvironment. Using a prototype bioreactor as shown in FIG. 2, which waspartially closed, no contamination was observed, despite being slightlyexposed to the external environment.

The bioreactor illustrated in FIGS. 1 and 2 may further include, butneed not necessarily include, positioning plates 28, 30 for positioningthe first and second substrates 14, 16, respectively, as well as coverplates 32, 34 for shielding various internal components of thebioreactor 10. Other plates, gaskets, fasteners, etc., may be included,as will be apparent to a skilled artisan. An optical load sensor couldalso be used in the bioreactor itself to help measure forces andfacilitate removal of the graft.

The bioreactor 10 described herein was designed for the fabrication ofscaffold-free bone-ligament-bone (BLB) multi-phasic tissue constructs.However, other cell types can be used, particularly those that form aspontaneously delaminating tissue monolayer or those that are capable ofsubstrate controlled tissue monolayer delamination (e.g., muscle tissue,nerve tissue, etc.). Current cell culture bioreactors are typicallydesigned for non-adherent cell suspension or cell expansion, but itshould be understood that these devices do not provide the means ofcapturing and co-culturing delaminated monolayers of multiple tissuetypes required for the formation of multi-phasic tissue constructs, suchas BLB tissue constructs.

With continued reference to FIG. 1, at least one translation mechanism18 in the bioreactor 10 extends between the first substrate 14 and thesecond substrate 16 so that a first tissue construct of a first celltype cultured on the first substrate 14 can be translated to the secondsubstrate 16 to form a multi-phasic tissue construct having a secondtissue construct of the second cell type cultured with the first tissueconstruct of the first cell type. The translation mechanism 18 may bemanual or automatic, so long as it is able to facilitate monolayercapture and transfer into co-culture. The translation mechanism mayinclude any structure or device capable of facilitating translation of atissue construct from one location in the bioreactor to anotherlocation. In the illustrated embodiment, the bioreactor 10 includes apair of sutures as the translation mechanism. The sutures may be usedfor translation. During translation, the sutures may be controlled bythe translation component 20, which is shown in an enlarged, explodedview in FIG. 3. In the translation component 20, magnets 36 move alonggrooves 38 to facilitate the lengthening or shortening of thetranslation mechanisms 18 as they are wound around anchoring spools 40.Manipulation of the position of the translation mechanisms 18 and theamount of tension may be used to control tissue construct length andgraft tension for delaminated tissue constructs. In a preferredembodiment, a three-dimensional, first tissue construct is captured onthe translation mechanisms 18 when the monolayer delaminates. The firsttissue construct may be allowed to mature on the translation mechanisms18, as the tissue contracts and reorganizes the extracellular matrix(ECM) structure while constrained under tension. This maturation phasemay occur over approximately two days if a bone tissue construct isformed; however, longer or shorter maturation times are possible. Tofacilitate translation and co-culture of the first tissue construct, thebioreactor 10 may be inverted. Then, in this embodiment, following therelease of tension in the translation mechanisms 18 by loosening theanchor spools 40, the first tissue construct can be translated to thesecond culture substrate 16 and tension may be re-established by pullingthe magnets 36 after the anchoring spools 40 have been retightened.

FIG. 4 is a top view of a bioreactor 10 that has a different embodimentfor the translation mechanism 18. In this embodiment, the translationmechanism 18 is a pin or a post, and more particularly, a pair of pinsin each of the two culture vessels 13. Since this translation mechanism18 is rigid, as opposed to the sutures of the previous embodiment, atranslation component that includes anchoring spools or other devicesmay not be necessary. As with the previous embodiment, the pins traversethe bioreactor 10, extending from the first substrate to the secondsubstrate. In a preferred embodiment, the pins are angled relative tothe substrates 14, 16. More particularly, the pins may be angled between45° and 80° relative to the first substrate, and preferably the pins areangled 70° relative to the first substrate. Using a steep angle mayallow the construct to translate faster. However, if the angle is toosteep, the translation mechanisms may not allow enough tissue to formaround the edges of the well, which may hinder contracting translation.In a further preferred embodiment, the pins are angled such that thedistance between them is greater at the first substrate than at thesecond substrate. This allows for a first construct to slide up andshrink until it reaches the second substrate during translation. In oneembodiment, after the first tissue construct reaches its position on thesecond substrate, the bioreactor 10 is flipped to seed cells on thesecond substrate that will delaminate and capture the first tissueconstruct, thereby forming a complex three-dimensional tissue constructor a multi-phasic tissue construct. The distance between the pins may befixed depending on the specifications of the graft. In the embodimentillustrated in FIG. 4, the pins are spaced to form two 15 mm boneconstructs in each culture vessel 13, and when the bioreactor 10 isflipped, a ligament tissue construct can form between the two boneconstructs. The ligament tissue construct is approximately 50 mm, whichgenerally equates to the distance between the two sets of pins.

With reference to FIGS. 5 and 6, in addition to angling pins relative tothe surfaces of the substrates 14, 16, one or more pins may be furtherangled relative to the sides 48 of the bioreactor. In other words, allof the pins may be angled relative to the y-axis of the plate toward thez-axis (e.g., by 70° relative to the surface of the first substrate 14),while the pins 18′ are further angled relative to the x-axis toward thez-axis (e.g., up to 90° from its prior position). This allows for thefirst tissue construct 42 to shrink as it is translated, resulting inthe first tissue construct 42′ at the second substrate afterdelamination. These angles are merely exemplary and may be altered.Further, sides 48 of the bioreactor 10 and the x- , y- , and z-axes areprovided as example reference points. Other reference points maycertainly be used.

FIG. 7 is an image of an assembly 70 that contains multiple bioreactors10. The bioreactors may be similarly designed, or may have differentdesigns, depending on the amount and/or types of tissue being formed.Further, as described above, the assembly 70 may be further equippedwith a perfusion system to help control the culture environment. With abioreactor assembly, several bioreactors may be connected in parallel toa single media reservoir and manifold system.

Preferably, the bioreactor 10 is fabricated from FDA compliantmaterials, and certain components of the bioreactor may be injectionmolded depending on the desired specifications of the bioreactor. Thesubstrates 14, 16 may be disposable cell culture surfaces, and the restof the bioreactor 10 may be sterilized using traditional sterilizationtechniques (e.g. autoclaving, ethylene oxide, etc.) and reused. FurtherUV treatment of the bioreactor 10 following assembly in a laminar flowhood, for example, can further ensure the sterility of the bioreactorprior to use. When fully closed, the bioreactor 10 can be used as acontainer for tissue preservation, storage, and shipping. Thus, themulti-phasic tissue construct can be preserved within the device so thatit can be stored and opened when needed by the surgeon.

In addition to the bioreactor 10 and bioreactor assembly 70, there isalso provided a method of forming a multi-phasic tissue construct in asingle culture chamber. The method may comprise the steps of forming afirst tissue construct in the culture chamber, adding cells to theculture chamber containing the first tissue construct, and culturing thecells to form a second tissue construct in the culture chamber. Thefirst tissue construct and the second tissue construct can be formedfrom different cell types and together comprise the multi-phasic tissueconstruct. In another implementation, which is further detailed belowwith respect to another embodiment of a bioreactor 110, there isprovided a method of forming a complex three-dimensional tissueconstruct in a single culture chamber. The method comprises the steps ofadding cells from a first cell source to a first substrate in theculture chamber, adding cells from a second cell source to a secondsubstrate in the culture chamber, and culturing the first cell sourceand the second cell source to form a first tissue construct and a secondtissue construct. The first and second tissue constructs are translatedvia one or more translation mechanisms to a co-culture zone between thefirst and second substrates, and the three-dimensional tissue constructis formed and has the second tissue construct of the second cell sourcecultured with the first tissue construct of the first cell source.

Prior art methods for complex three-dimensional and multi-phasic tissueconstruct formation require simultaneous culture of both tissueconstructs in separate culture chambers, and more particularly, BLBfabrication requires the simultaneous culture of bone and ligamentmonolayers in separate culture chambers. Forming the actual multi-phasicBLB graft requires the timed, manual transfer of two three-dimensionalbone constructs onto a confluent ligament monolayer prior to themonolayer's delamination. The delicate timing and difficulty associatedwith manually performing this process can make it difficult to replicatein a closed bioreactor design. The sequential preparation of two tissuetypes such as bone and ligament tissues, in accordance with the methodsdescribed herein, would greatly eliminate the technical complexities ofthis process and simplify the bioreactor design. Thus, the ability toform a confluent, spontaneously delaminating ligament monolayer from asingle fibrogenic cell suspension while in the presence of pre-formedbone constructs needed to be evaluated.

In order to test the viability of the methodology, the process offorming a single three-dimensional BLB construct of suitable diameterfor use as a graft in ACL reconstruction in a single culture chamber wasevaluated. Bone marrow was aspirated from the iliac crest of sheep usinga Monoject Illinois needle (Sherwood Medical Company, St. Louis, Mo.)with the animal under general anesthesia induced by intravenous propofoland sustained with inhalation of isoflurane in oxygen. An exemplaryminimum of 15 mL of bone marrow volume was collected using heparinizedneedles and dispensed into ethylenediaminetetraacetic acid (EDTA) bloodcollection tubes (BD, San Jose, Calif.) for processing. The marrow wasfiltered through a 110 mm filter to remove solid debris and consolidatedinto a single 50 mL conical. An equivalent volume of Dulbecco'sPhosphate-Buffered Saline (DPBS) pH 7.2 (Gibco BRL Cat#14190-144) wasadded. The marrow solution was slowly added to a 15 mL layer ofFicoll-Paque Premium (GE Healthcare, Munich, Germany) contained within aseparate conical and centrifuged (AccuSpin FR; Beckman Coulter Inc.,Fullerton, Calif.) at 600 g for 30 minutes @25C. The upper layercontaining plasma and platelets was discarded and mononuclear celllayers, containing the mesenchymal stem cell (MSC) population, weretransferred into a new sterile conical. The cells were suspended in atleast three times their volume of DPBS and centrifuged at 500 g for 10minutes. The supernatant was removed and an equivalent volume ofammonium-chloride-potassium (ACK) lysis buffer was added to the pelletvolume, lysing any remaining red blood cells. The conical was filledwith DPBS and centrifuged at 400 g for 5 min. The supernatant was againremoved and the MSC pellet was re-suspended in 20 mL of growth media(GM), which in this embodiment was Dulbecco's Modified Eagle Medium(DMEM) supplemented with 20% fetal bovine serum (FBS) and 2% fungizone(ABAM). Cells were counted utilizing a Countess automated countingsystem and plated at a density of 40 k cells/cm² in a 150 mm tissueculture dish.

The adherent cell population was allowed to attach for 3 days before theplate was rinsed with DPBS to remove any remaining debris orcontaminating non-adherent cell populations. Following plating, cellswere immediately cultured towards osteogenic or ligamentous lineages.The osteogenic growth medium consisted of GM supplemented with 600 ng/mLbasic fibroblast growth factor (bFGF; Peprotech), 400 ng/mldexamethasone (dex; Sigma-Aldrich). The ligamentous growth mediaconsisted of GM supplemented with only bFGF. Plates were fed respectivebone and ligament media every other day. At approximately 50% confluencethe plate-adherent MSC populations were trypsinized (trypsin-0.25%EDTA), combined into a single conical, and passaged at a density of 5 kcells/cm² every other day. Following passage three, ligamentous derivedMSCs (ligament cells) were trypsinized and cryogenically preserved. MSCsinduced to a osteogenic lineage (bone cells) were passaged andcryopreserved after passage four. Cells were cryogenically preserved ata concentration of 5×10⁶ cells/mL GM plus 10% dimethyl sulfoxide (DMSO)(Sigma-Aldrige). One mL volumes were aliquoted into 2mL cryovials(Nalgene) and slowly frozen (approximately 1 C/hr) overnight to −80C.The vial was then transferred into a liquid nitrogen freezer forlong-term storage.

As needed, cells were removed from liquid nitrogen storage and placed ina 37 C water bath. After the media turned to liquid, the entire volumeof cells and media was transferred to a sterile 15 mL conical containingat least 3 times their volume of warmed GM. The cell suspension wascentrifuged at 1500 RPM for 5 min at 37 C. The supernatant was aspiratedand remaining cells were re-suspended in fresh GM and counted. Cellviability was assessed with trypan blue dye (Life Technologies).Recovered cells utilized for tissue formation contained greater than 90%viable cells. Cells were seeded onto sterile polystyrene tissue cultureplates at a viable cell density of 21 k cells/cm². Bone plates were fedGM supplemented with 400 ng/mL dex, 13 mg/mL ascorbic acid, 5 mg/mLL-proline, and 600 ng/mL bFGF. The ligament medium was similar to theGM, lacking only the dex.

With reference to FIG. 8, monolayers derived from both bone and ligamentcryopreserved MSC lineages successfully formed and spontaneouslydelaminated, demonstrating the ability of cryogenically preserved cellsto form BLB grafts. Additionally, seeding of ligament cells followingtransfer of bone constructs to the culture surface did not interferewith ligament monolayer formation required for successful co-culture andBLB formation. The delaminating ligament monolayers fully surrounded thebone constructs capturing them within the newly formed BLB construct.The diameter of the ligament region was noticeably thin (0.3±0.1 mm,n=5) with about half of the constructs failing in the ligamentmid-substance after 2 weeks. The results demonstrate the ability to forma BLB sequentially in the same dish by first adding bone constructsbefore seeding ligament cells. Thus, as shown FIG. 8, a first tissueconstruct 42 of a first tissue type was successfully formed with asecond tissue construct 44 of a second tissue type, thereby forming amulti-phasic tissue construct 46.

FIGS. 9 and 10 show other multi-phasic tissue constructs 46 that wereformed in accordance with the methods described herein. Larger pinplates were fabricated utilizing 150 mm tissue culture plates, with aculture surface area of 182 cm². The plates included two metal pins orposts located 50 mm apart, which were centered and aligned uni-axially.As a control, 110 mm pin plates, with a culture surface area of 82 cm²,were also fabricated. Pin locations were the same in each of the plates.Plates were UV treated for at least 1 hour and sprayed with 70% ethylalcohol (ETOH) and rinsed with approximately 20 mL DPBS prior to use.Ligament cells were seeded onto 110 mm and 150 mm pin plates, grown intoa confluent monolayer, and spontaneously delaminated into a threedimensional tissue construct. Construct diameters were compared at aone-week time point following delamination. FIG. 9 shows that the 110 mmpin-plate formed a construct approximately 2 mm in diameter (n=3), whileFIG. 10 shows that constructs fabricated in the 150 mm pin-plates formeda construct about 5 mm in diameter (n=3).

Adding MSCs to individual wells containing strategically placed,pre-formed three-dimensional tissue constructs of a first tissue type,formed a multi-phasic construct. This is in sharp contrast to previousmethodologies that required combination of three-dimensional tissueconstructs of a first tissue type onto confluent monolayers of a secondtissue type to form the multi-phasic tissue. The results demonstrate thefeasibility to form a multi-phasic tissue construct, such as a BLBtissue construct, within a single well by sequentially seedingcryogenically preserved bone and ligament cells, for example. Thisprocess adaptation eliminates the need for technicians to combine boneand ligament tissues to form a BLB in this example. Cells, as opposed tothree dimensional tissue constructs, can be added into the system,lending itself nicely for injection into a closed system. Elimination oftechnician dependent steps allows for the process to be automated andperformed in a closed system, which is desirable for use within abioreactor such as bioreactor 10.

The use of cryogenically preserved allogeneic cells can allow forlarge-scale expansion of cells that can be well characterized and usedas needed for graft fabrication. This is a significant advantage forprocess scalability compared to fresh cells, as the starting cellpopulation can be screened and evaluated before investing time andresources into large volume construct production. From a regulatoryperspective, a well-defined starting cell population with specificacceptance criteria may be necessary. Allogeneic cells have theadvantage of performing this characterization only once. Autogenic cellsmust be characterized for each donor and the resulting tissue constructsare patient specific. This can significantly increase the economy ofscale and limit commercialization potential.

Additionally, the elimination of larger diameter grafts formed on the150 mm plates compared to 110 mm plates, as shown in FIGS. 9 and 10,reveal the ability to control graft size through the manipulation ofculture surface area. This process eliminates the need for manual fusionof multiple constructs by a technician, again, a technician-dependentstep that can impede the development of efficient, automatedmanufacturing processes, thereby making the process suitable forincorporation with a bioreactor. The change of construct diameter,passive construct tension, and tissue mechanics should be evaluatedfurther. The described results demonstrate protocol adaptations thatwere used to facilitate fabrication of a BLB construct within singlewell, closed, bioreactor system.

As alluded to above, the methods described herein may also beadministered in a bioreactor, such as bioreactor 10. FIGS. 11A-11Eschematically illustrate one embodiment of a method of forming a complexthree-dimensional or multi-phasic tissue construct. While thisembodiment of the method is shown as being performed with one particulardesign for the bioreactor 10, it should be understood that the method isnot limited to use with the illustrated bioreactor, as any suitablebioreactor may be employed, such as bioreactor 10 shown in FIGS. 4-7 inthis example. Method adjustments can be made for other bioreactorimplementations, such as the bioreactor 110 depicted in FIG. 12, whichis described in further detail below. Moreover, while the descriptionbelow is provided with reference to the formation of a BLB multi-phasictissue construct, other tissue types may be used and/or formed, such asthose that form a spontaneously delaminating tissue monolayer or thosethat are capable of substrate controlled tissue monolayer delamination(e.g., muscle tissue, nerve tissue, etc.).

FIGS. 11A-11E are schematic side views of bioreactor 10 having a firstsubstrate 14 for forming a first tissue construct of a first cell type,and a second substrate 16 for forming a second tissue construct of asecond cell type, which in this embodiment, is ligament tissue constructspanning two bone tissue constructs (i.e., a BLB multi-phasic tissueconstruct). Preferably, if forming a BLB construct, the bioreactor iskept at 37 C, 95% humidity and 5% CO₂ to allow for cell growth, withmedia exchange occurring within a sterile laminar flow hood. Withreference to FIG. 11A, during bone monolayer formation, MSCs are seededonto the first substrate 14, which preferably comprises a sterilepolystyrene culture surface coated with or without cell adhesionproteins such as laminin. The MSCs may be induced towards an osteogeniclineage and sterilely delivered into individual culture vessels 13within the culture chamber 12 of the bioreactor 10 and fed nutrients andgrowth factors specific to bone formation. Media exchange may becontrolled manually with sterile injection of media in anopen-environment or automatically with perfused media. MSCs of the bonemonolayer growth process may be seeded at an initial density ofapproximately 21,000 cells/cm². Seeding density may not be critical tothe success of forming a multi-phasic tissue construct, as it may onlyaffect the rate of monolayer formation.

With reference to FIG. 11B, after seeding, cells proliferate forming amonolayer, and differentiate towards an osteogenic lineage. Whileadditional serum-containing cell culture media and growth factors may beused, a preferred growth medium to optimize the cell proliferation andpromote robust osteogenic ECM production consists of DMEM supplementedwith 20% fetal bovine serum and antibiotics along with the addition ofdex, fibroblast growth factor (FGF-b), and ascorbic acid/L-proline.Growth conditions that are preferable include a pH of about 7.4, amedium temperature of 37 C, and a sterile air environment consisting ofup to 20% CO₂. Media should be exchanged at a sufficient rate and volumeto prevent depletion of critical media constituents by the growingcells, with preferably 50% of the media volume being replaced every twodays.

Following sufficient ECM matrix production by the seeded cells, aconfluent bone monolayer is formed on the first substrate 14, which inone embodiment, occurred approximately 5 days at the described seedingdensity. Growth-promoting medium can be replaced with differentiationmedium consisting of DMEM, supplemented with 7% horse serum, antibiotic,transforming growth factor-beta (TGF-b), ascorbic acid/L-proline, anddex to induce cell differentiation, integration and passive tension thatleads to subsequent spontaneous delamination of a confluent tissuemonolayer. Bone monolayer delamination within the culture vessel 12 maybe captured on the translation mechanisms 18, forming a threedimensional bone tissue construct 42 as shown in FIG. 11B. The bonetissue constructs 42 may be allowed to mature on the translationmechanisms 18 as the tissue continues to contract and reorganize the ECMstructure while constrained under tension. This maturation phase canoccur over approximately 2 days; however, longer or shorter maturationtimes are possible. To facilitate translation and co-culture of the boneconstructs 42, the bioreactor 10 may be inverted or flipped, as shown inFIG. 11C. Bioreactor inversion, and possibly the release of tension inthe sutures depending on the particularities of the bioreactor beingused, result in the translation of the bone tissue 42 to the secondsubstrate or ligament substrate 16, as shown in FIG. 11D. Bioreactorinversion may be done by hand, by a technician for example, or it may beautomated by a mechanical device. If a bioreactor assembly is beingused, such as the bioreactor assembly 70 shown in FIG. 7, the entireassembly may be inverted or the assembly may be designed such that thebioreactors are individually inverted at various times.

Following translation of the bone constructs 42, MSCs are seeded ontothe second substrate 16 at an initial density of approximately 21,000cells/cm² in one example. The osteogenic medium may be replaced withligament growth medium to promote fibroblastic differentiation andproduction of ligamentous ECM. This process can require approximately 8days in culture. The ligamentous medium consists of DMEM supplementedwith 20% fetal bovine serum and antibiotics along with the addition ofFGF-b, and ascorbic acid/L-proline. Ligament medium may be exchanged at50% of volume every 2 days. Other environmental conditions required forligament tissue growth may be the same as for bone tissue growth.

Following formation of a ligament monolayer upon the ligament culturesubstrate 16, the medium may be switched to differentiation medium tofurther induce matrix production and cell-matrix tension. Theligamentous differentiation medium may be composed of DMEM, supplementedwith 7% horse serum, antibiotic, transforming growth factor-beta(TGF-b), and ascorbic acid/L-proline. As the ligament monolayerspontaneously delaminates forming the ligament tissue structure 44, thetranslation mechanisms 18 can capture it, surrounding the boneconstructs 42 on the second substrate 16. The end result is amulti-phasic BLB construct 46 that may continue to mature (remodel,condense, and develop ECM) over time in culture, as shown in FIG. 11E.This maturation phase may require approximately 1 week, although shorterand longer periods are possible.

The resulting BLB construct 46 can be grafted into the patient at thispoint or if desired, further conditioned mechanically through thephysical manipulation of the translation mechanisms 18, which aresutures in this embodiment. If implanted, the incorporated sutures maybe utilized to secure the graft into the patient during the surgicalprocedure. This may be desirable for trans-osseous rotator cuff repairor anatomical ACL reconstructive surgery, where the multi-phasic tissueconstruct is sutured to the periosteum of the bone. If sutures areincluded during fabrication of the complex three-dimensional tissueconstruct, it is desirable if the sutures provide adequate material tosecure the ends of the graft and anchor the multi-phasic tissueconstruct during surgery. Alternative suture configurations may beincluded to enhance suture-graft integration and provide additionalstability during implantation and fixation to native tissue. It ispreferable that a fresh BLB construct be used within 3 weeks followingformation, as cells and appropriate graft tension are difficult tomaintain in culture for extended periods of time. Alternatively,freezing, acellularizing, or other means of preservation may be used topreserve the construct.

The resulting multi-phasic tissue construct 46 was continuous, havingcompositionally different tissue at each end. A mineralized matrix wasobserved in the bone regions 42 of the graft despite the removal ofdexamethasone and prolonged exposure to ligament growth medium. Further,it should be understood that the size of the multi-phasic tissueconstruct formed in a bioreactor may be slightly smaller than ifproduced according to other methods (e.g., 5 mm and 2 mm in diameter for110 mm and 150 mm plates, respectively, versus 2-3 mm diameter for thebioreactor). However, the resulting tissue construct will likely grow insize in vivo to approximate the native tissue dimensions. Despite this,the in vitro construct should still maintain a consistent and robustdiameter to provide sufficient structural integrity during theimplantation procedure. Thus, strategies to maintain the multi-phasictissue construct size, such as altering culture surface area, removingTGF-B, or releasing suture tension, for example, may be used to preventexcessive tissue condensation and better preserve construct diameter andviability. It is likely that inherent variability in cell ECM synthesisand remodeling was a factor in the size disparity.

FIGS. 12-17 illustrate another embodiment of a bioreactor 110. Thebioreactor 110 has an open-ended design and can be implemented in anumber of different ways. The bioreactor 110 includes a body 111 thatgenerally defines a culture chamber 112. The body 111 in this embodimentincludes a first wall 113, a second wall 115, and a third wall 117,which are shown in FIGS. 12 and 13A. FIG. 13B illustrates a side view ofthe body 111 showing the first wall 113. The first wall 113 includes afirst substrate 114; the second wall 112 includes a second substrate116; and the third wall 117 includes a third substrate 119. Thesubstrates 114, 116, 119 are removable and/or disposable plates in thisembodiment, which can be employed with a reusable body. Alternatively,the substrates 114, 116, 119 may be integrally formed with the walls113, 115, 117 of the body 111 or have another operable configuration.FIG. 12 shows an open end 121, and there is also a closed end 123opposite the open end 121. Openings 125 within the interior of the body111 are designed to mate with a cap 127, which is illustrated in FIGS.14A and 14B. The illustrated embodiment includes three substrates 114,116, 119 and three walls 113, 115, 117 which are arranged to form apolygonal-shaped culture chamber 112. The three substrates and wallsform a triangle; however, it is possible to include more substrates andwalls. For example, bioreactor bodies that includes four, five, six,seven, eight, or more than eight walls, to cite a few examples, arecertainly possible. The number of substrates for culturing cell sourcesis preferably between one and eight. With more substrates, more complexshaped tissue constructs can be created, and larger volumes of tissuescan be fabricated. A polygonal-shaped body, such as that shown in thefigures, can be optimized to reduce the amount of media used in theculture chamber 112, which may lower the cost of culturing cell sources.The bioreactor 110 or its various components may be formed of aninjection molded plastic to help reduce cost and encourage scalability,by assembling a number of bioreactors to a single manifold for feeding.

The bioreactor 110 may include a cap 127 to mate with the open end 121of the body 111 to form a closed, sterile environment for cellculturing, as shown in FIGS. 14A and 14B. The cap 127 can have capopenings 129 which are designed to mate with the openings 125 of thebody 111. Having the geometry of the cap 127 match or mimic the geometryof the body 111 can help reduce the amount of media needed. The cap 127may have other features such as a handle 131 to help with placement andremoval. It may also be possible to have a bioreactor with two open endsand a cap situated at either end. Silicone glue or vacuum grease may beused to help keep the system water and airtight. The body 111, the cap127, and/or the substrate plates 114, 116, 119 may be formed of atransparent plastic material which allows a user to easily see cellgrowth on the substrates, when the tissue constructs translate and fusein a desired position, and the final construct formation. Other featuresor components may be included with the body 111 and/or the cap 127besides those illustrated in the figures. For example, a valve systemmay be used to help simultaneously grow and feed multiple tissue typeswith different types of media. One or more pressure release holes may beincluded for the displacement of gas when media is inserted into theculture chamber. Ports for the infusion and aspiration of cells andmedia that allow for near-complete aspiration of the media during mediachanges may be added. In one embodiment, corners of the bioreactor canbe equipped with gates that regulate the flow of media between cultureareas, thereby allowing for multiple types of media to be fedsimultaneously to different cell monolayers.

With reference to FIGS. 12 and 15, bioreactor 110 includes one or moretranslation mechanisms 118. Preferably, each substrate 114, 116, 119 isequipped with translation mechanisms 118 in the form of pins whichextend from a culture surface 135 toward a co-culture zone 133. In thisembodiment, the co-culture zone 133 is centrally located in the culturechamber 112 between the three substrates 114, 116, 119, with translationmechanisms 118 extending from each respective substrate to theco-culture zone 133. In this embodiment, the co-culture zone is locatedremote from the substrates in the central portion of the bioreactor body111; however, other locations for the co-culture zone are certainlypossible. FIG. 15 shows translation mechanisms 118 in the form of a pairof angled pins, which may be similar to the embodiment shown anddescribed with respect to FIGS. 4-6. More pins or translation mechanisms118 may be included with each substrate, and each substrate may have adifferent configuration of translation mechanisms. Moreover, eachsubstrate may include divots or openings to allow for insertion of pinsin a number of locations. Divots may be preferred over holes in someembodiments, to help preserve the integrity of the surface 135 which maybe formed of plasma treated polystyrene plastic. Additionally, eachsubstrate can have a different size or geometry. For example, physicalbarriers such as multiple wells or a spatially optimized Teflon coatingmay be used. Other features such as a tab to assist with insertion andremoval are certainly possible as well.

FIGS. 16A-16F illustrate various implementations for a substrate, suchas substrate 114 with cell culture surface 135. In one embodiment, thesubstrate 114 is made from a plasma treated tissue plastic such aspolystyrene. Plasma treated tissue plastic is used to allow the cells toadhere and proliferate to form a monolayer. The plastic sheets can becoated with either non-adherent coatings (e.g., silicone, Teflon, etc.),or adherent coatings (e.g., adhesion proteins, Matrigel, etc.).Different growth factors may also be used to coat the surface 135 toenhance the specificity of cell growth. The coating type andconfiguration can be manipulated to change the shape or cell type foradhesion to the surface of the plastic. FIG. 16A illustrates a plaincell culture surface 135. In FIG. 16B, the outer white areas are coatedwith Teflon. Because the cells will not adhere to Teflon, the coating onthe cell surface 135 of this embodiment may be used to decrease themonolayer size. FIGS. 16C and 16D are similar to FIG. 16B, with an outerregion coated in Teflon, but in these embodiments, the shape of thecoating is altered to change the shape and size of the tissue constructto be formed. FIG. 16E includes a culture surface 135 that is coatedwith adhesion proteins to increase cell adhesion to adhere specific celltypes. FIG. 16F shows a culture surface 135 that is coated with growthfactors to enhance the growth of specific cells adherent to thesubstrate 114. Adhesion proteins and growth factors can be used alone orin combination to adhere and enhance the growth of adherent cells.Moreover, the bioreactor can include a number of different substrateshaving different coating configurations for each desired tissueconstruct to be formed, which ultimately impacts the multi-phasic orcomplex three-dimensional tissue construct.

FIG. 17 is a schematic illustration of the bioreactor 110. Thetranslation mechanisms 118 in this embodiment are pins which are angledfrom 50 to 80 degrees, and preferably 70 degrees. In this embodiment,passive tension of each construct 142, 144 allows for the translation ofeach construct up each pin set toward the co-culture zone 133. Theco-culture zone 133 can include interfacing or touching translationmechanisms 118, or the translation mechanisms may be spaced from eachother to a certain extent. At the co-culture zone 133, the constructs142, 144 fuse to form a complex three-dimensional tissue construct 146.A complex three-dimensional tissue construct includes two or more tissueconstructs cultured from two or more cell sources. The cell sources mayinclude the same cell type. In another embodiment, the complexthree-dimensional tissue construct is a multi-phasic tissue constructwhich includes two or more tissue constructs cultured from two or morecell sources, with the cell sources having one or more different typesof cells, such as the BLB construct described above. Applications of theformed constructs include repair of rotator cuff or anterior cruciateligament injuries, to cite a few examples.

FIGS. 18 and 19 schematically represent various complexthree-dimensional tissue constructs 146 that may be formed with thebioreactor 110. In FIG. 18, three tissue constructs 142, 144, 145translate to the co-culture zone 133 to form a complex three-dimensionaltissue construct 146 which is in column form. In FIG. 19, the threetissue constructs 142, 144, 145 may be formed with translationmechanisms 118 having particular configurations so as to allowtranslation of about one half of each construct to the co-culture zone.The translation mechanisms 118 may include hooks at the end to help stopthe translation of the tissue construct at a certain point. Theresulting complex three-dimensional tissue construct 146 has a threepronged configuration that may be used as a rotator cuff graft, forexample.

Other optional components for the bioreactor 110 are illustrated inFIGS. 20A, 20B, and 21, such as a rotary device 151 which allows thebioreactor 110 to be rotated about a rotary axis 153, and a grabbingdevice 155 which can help facilitate removal or insertion of thesubstrates 114, 116, 119. The rotary device 151 shown in FIGS. 20A and20B can help allow for easy cell seeding, adherence of cells to thedifferent substrates, and media changes. The grabbing device 155depicted in FIG. 21 may be three-pronged as shown, or have acorresponding number of prongs depending on the number of walls and/orsubstrates of the bioreactor. All of the substrates 114, 116, 119 shouldbe removed at the same time to avoid tearing the complexthree-dimensional tissue construct. The grabbing device 155 can helpgrab and remove all three substrates at one time to harvest the tissuefrom the culture chamber 112.

FIGS. 22-25 illustrate various procedures that may be used to evaluatevarious qualities of a formed three-dimensional tissue construct. Forthe various exemplary procedures, a BLB multi-phasic tissue constructwas removed from the bioreactor 10 and subjected to further evaluation.More particularly, separate parts of the construct were allocated formechanical, histological, and molecular biology analysis. Approximatelyhalf of the bone/ligament construct was taken for histology andmolecular biology testing. The rest was attached to a custom builttensile tester and submerged in DPBS for subsequent tensile testing. Thetensile testing set-up can be seen in FIG. 22, which is an image of thedescribed testing set-up of the ligament region 44 of the BLB construct46. The missing bone region on the right, shown by the lack of sutures,indicates the region removed for PCR analysis. The construct was securedusing custom Velcro grips and incrementally lengthened to approximatethe original length within the bioreactor. Sixty-micron beads werepainted onto the surface of the tissue for post analysis digital imagecorrelation to extrapolate the tissue-level strain during testing. Theconstruct 46 was pulled until failure at a rate of 0.01 strain/sec. Theload and strain at failure was recorded.

FIG. 23 is a graph showing stress-strain data of multi-phasic tissueconstructs formed in accordance with various embodiments. Referencenumerals 54, 56, 58 represent manually fabricated multi-phasic tissueconstructs, and reference numerals 60, 62 represent bioreactorfabricated multi-phasic tissue constructs. Ligament constructs formedwith a manual process, had a modulus of 0.38±0.12 MPa (n=3) at aphysiological strain range of 0.05-0.07. The bioreactor constructs weresimilar, with an average tissue modulus of 0.28±0.09 MPa (n=2). Themechanics of the bone regions were not evaluated. The relative standarddeviation within each group was approximately 30%. The diameter of thebioreactor constructs were 1.55±0.71 mm for BLBs (n=2) and 0.91±0.03 forligament only constructs captured on pin plates (n=3).

For histological staining analysis, as shown in FIGS. 24A-24F, unfixedsamples were placed into TBS medium (Triangle Biological Sciences),frozen in cold isopentane, and stored at −80 C until needed. Fixedsamples were placed in 4% paraformaldehyde (PFA) for 1 hour. The samplewas then placed into 15% sucrose solution for 1 hr at 4 C and thenovernight (>12 hrs) in 30% sucrose solution also at 4 C. The constructswere then transferred into TBS medium, frozen over dry ice, and storedat −80 C until needed. Samples were cut to obtain cross and longitudinalsections with a cryostat at a thickness of 12 μm, adhered to SuperfrostPlus microscopy slides and used for staining. Sections were stained forgeneral morphology observations with hematoxylin and eosin (H&E).Mineral content of the tissue was determined via alizarin red staining(Sigma), and collagen with picro-sirius red stain. Alizarin red stainingshows mineral nodules within the bone regions of the graft and absentfrom the ligament region of the graft, evidence of a bi-modal tissuedistribution within the BLB graft, as shown in FIGS. 24A-24C.Mineralization was comparable to bone and ligament constructs culutredseperately in ostogenic and fibrogenic media respecivly, demonstrating acapacity of the tissue to maintain its differntiated state while inco-culutre. The construct was composed of primarily type 1 collagen andcontained viable nuclei thoughout, as shown in FIGS. 24D-24F.

With reference to FIG. 25, Y-chromosome PCR analysis was also performed.Genomic DNA was isolated from respective bone and ligament regions ofthe BLB tissue constructs by proteinase K digestion followed by ethanolprecipitation. A PCR based assay for the ovine-specific Y-chromosomerepeat sequence Ucd043 was used to determine the presence of male cellsin BLB explant samples. Duplex PCR was performed usingSCUcd043.FWD/SCUcd043. REV primers to amplify Ucd043 together withP1-5EZ/P2-3EZ primers to amplify the ZFY/ZFX locus. P1-5EZ and P2-3EZprimers also provided an internal control for amplification. Sensitivityof the assay was assessed with a dilution panel of 110 pg, 25 pg, and5pg of male ovine DNA. Subsequent experimental reactions were carriedout with 25 ng of genomic DNA template. The PCR analysis demonstratesco-culture of multiple tissues within a continuous multi-phasic tissueconstruct. The male Y chromosome was detected in only bone regions ofthe construct, as shown in FIG. 25. Female tissue was detectedthroughout the construct and most highly expressed in central ligamentregion. Both the bone and ligament regions contained significant amountsof DNA.

It is to be understood that the foregoing description is of one or morepreferred exemplary embodiments of the invention. The invention is notlimited to the particular embodiment(s) disclosed herein, but rather isdefined solely by the claims below. Furthermore, the statementscontained in the foregoing description relate to particular embodimentsand are not to be construed as limitations on the scope of the inventionor on the definition of terms used in the claims, except where a term orphrase is expressly defined above. Various other embodiments and variouschanges and modifications to the disclosed embodiment(s) will becomeapparent to those skilled in the art. All such other embodiments,changes, and modifications are intended to come within the scope of theappended claims.

As used in this specification and claims, the terms “for example,”“e.g.,” “for instance,” and “such as,” and the verbs “comprising,”“having,” “including,” and their other verb forms, when used inconjunction with a listing of one or more components or other items, areeach to be construed as open-ended, meaning that the listing is not tobe considered as excluding other, additional components or items. Otherterms are to be construed using their broadest reasonable meaning unlessthey are used in a context that requires a different interpretation.

1. A bioreactor for forming a complex three-dimensional tissueconstruct, comprising: a first substrate for culturing a first cellsource; a second substrate for culturing a second cell source, whereinthe first substrate and the second substrate comprise two walls of aculture chamber; and at least one translation mechanism in the culturechamber that at least partially extends between the first substrate andthe second substrate so that a first tissue construct of the first cellsource cultured on the first substrate or a second tissue construct ofthe second cell source cultured on the second substrate can betranslated via the translation mechanism to form the complexthree-dimensional tissue construct having the second tissue construct ofthe second cell source cultured with the first tissue construct of thefirst cell source.
 2. The bioreactor of claim 1, wherein the firstsubstrate opposes the second substrate so that when the bioreactor isinverted, the first tissue construct cultured on the first substrate canbe translated to the second substrate.
 3. The bioreactor of claim 1,wherein the culture chamber includes multiple culture vessels separatedby one or more non-adherent barriers.
 4. The bioreactor of claim 3,wherein a single media reservoir is used for all of the first substratesand another single media reservoir is used for all of the secondsubstrates.
 5. The bioreactor of claim 1, wherein the first and secondsubstrates facilitate adherent cell culturing.
 6. The bioreactor ofclaim 1, further comprising one or more ports for exchanging differenttypes of culture media, each of the ports providing fluidiccommunication from the culture chamber to a location external to thebioreactor so as to enable the introduction of the culture media intothe culture chamber.
 7. The bioreactor of claim 1, further comprising aperfusion system for temperature control and/or fluid exchange.
 8. Thebioreactor of claim 1, wherein the at least one translation mechanismcomprises at least one suture that can be used to secure the complexthree-dimensional tissue construct during implantation.
 9. Thebioreactor of claim 1, wherein the at least one translation mechanismcomprises at least one pin.
 10. The bioreactor of claim 9, wherein theat least one pin is angled relative to a surface of one of thesubstrates.
 11. The bioreactor of claim 1, wherein the at least onetranslation mechanism comprises two pins, wherein both pins are angledsuch that the distance between them is greater at the first substratethan at the second substrate.
 12. The bioreactor of claim 1, wherein atleast one translation mechanism extends from the first substrate to meeta second translation mechanism extending from the second substrate at aco-culture zone located between the translation mechanisms of the firstand second substrates.
 13. The bioreactor of claim 12, furthercomprising a third substrate for culturing a third cell source and athird translation mechanism extending from the third substrate to theco-culture zone.
 14. The bioreactor of claim 13, comprising three ormore substrates, wherein the three or more substrates comprise walls ofa polygonal-shaped culture chamber.
 15. The bioreactor of claim 1,wherein the first cell source includes cells of a first cell type andthe second cell source includes cells of a second cell type, and thethree-dimensional tissue construct is a multi-phasic tissue construct.16. A method of forming a multi-phasic tissue construct in a singleculture chamber, comprising the steps of: forming a first tissueconstruct in the culture chamber; adding cells to the culture chambercontaining the first tissue construct; and culturing the cells to form asecond tissue construct in the culture chamber, wherein the first tissueconstruct and the second tissue construct are formed from different celltypes and together comprise the multi-phasic tissue construct.
 17. Themethod of claim 16, wherein the cells are allogeneic mesenchymal sterncells (MSCs).
 18. The method of claim 16, further comprising the step ofinverting the culture chamber containing the first tissue constructbefore adding the cells to the culture chamber.
 19. The method of claim16, wherein the first tissue construct is a bone tissue construct, thesecond tissue construct is a ligament tissue construct, and themulti-phasic tissue construct is a bone-ligament tissue construct, 20.The method of claim 19, wherein the culture chamber is at leastpartially divided into two culture vessels, and two bone tissueconstructs are formed in each culture vessel of the culture chamber, andthe ligament tissue construct extends between the two bone tissueconstructs so that the multi-phasic tissue construct is abone-ligament-bone (BLB) tissue construct.
 21. A method of forming acomplex three-dimensional tissue construct in a single culture chamber,comprising the steps of: adding cells from a first cell source to afirst substrate in the culture chamber; adding cells from a second cellsource to a second substrate in the culture chamber; culturing the firstcell source and the second cell source to form a first tissue constructand a second tissue construct; translating the first and second tissueconstructs via one or more translation mechanisms to a co-culture zonebetween the first and second substrates; and forming the complexthree-dimensional tissue construct having the second tissue construct ofthe second cell source cultured with the first tissue construct of thefirst cell source.
 22. The method of claim 21, further comprising thestep of adding cells from a third cell source to a third substrate inthe culture chamber and culturing the cells of the third cell sourcewith the first cell source and the second cell source to form the firsttissue construct, the second tissue construct, and a third tissueconstruct, wherein the translating step includes translating the thirdtissue construct via the one or more translation mechanisms to theco-culture zone that is between the first, second, and third substrates,and forming the complex three-dimensional tissue construct having thethird tissue construct of the third cell source cultured with the firstand second tissue constructs of the first and second cell sources.